The present invention is directed to a gene and gene product of sweetgum (Liquidambar styraciflua), referred to herein as the LSAG gene, which is a homolog of the AGAMOUS gene (Meyerowitz et al. (1998)). The LSAG gene is a flowering gene which is required for normal flower development and sweetgum fertility. The present invention is also directed to an LSSAG promoter and/or enhancer and/or intron which can be used to prepare gene constructs and transgenic plants for tissue specific expression of the gene construct. The gene constructs and transgenic plants are further aspects of the present invention. Finally, the present invention is directed to methods to control flower development and hence assure sterility in transgenic sweetgum.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
In order for transgenic plants to be grown outdoors, it is necessary that they not be harmful to the environment. For forest trees, one criterion is that they be reproductively sterile: unable to form flowers or gametes, and therefore unable to disperse pollen or seeds containing transgenes. This can be achieved by interfering with the function of genes required for normal reproductive development, as has been demonstrated by numerous mutations in Arabidopsis and other model species. However, the interference with such genes must not interfere with normal vegetative growth, especially in trees planted for production of timber or pulp.
There are many possible genes that can be used to control flowering. Research during the past two decades on several herbaceous species, especially the model species Arabidopsis, has produced a wealth of information about genes that are involved in the development of flowers (Levy and Dean (1998)). Although literally thousands of genes are activated during floral development, studies have focused on a few dozen xe2x80x9cmaster controlxe2x80x9d genes that regulate transcription during the process. These genes were often first detected by the fact that mutations that inactivated them yielded grossly deformed flowers, or occasionally no flowers at all. The Arabidopsis gene AGAMOUS (AG) codes for a DNA-binding protein required for normal development of stamens and carpels. When it is mutated, petals are formed in the place of stamens and the carpels are replaced by nested sterile flowers. In situ hybridization studies have shown that the gene is transcribed only in floral meristems and primordia for stamens and carpels. The AGAMOUS protein shows sequence similarity with other regulatory proteins in the so-called MADS-box domains.
The methods of interfering with gene function in a transgenic plant include introducing a synthetic gene that causes sense or antisense suppression of the target gene (Taylor and Jorgensen (1992)). The suppression methods require substantial similarity between the target gene and the suppressing gene, greater than 80% nucleotide sequence identity (Mol et al. (1995)). Sweetgum is not closely related to the model plant systems from which most flowering genes have been isolated, and thus there do not seem to be any sequences that can reliably be used to suppress flower development in this species.
A gene encoding a dominant negative form of a protein essential for flowering may also be used to create a sterile plant. This has been reported in Arabidopsis for AGAMOUS (Mizukami et al. (1996)), but it is not known whether the dominant negative AGAMOUS gene will produce the same result in a heterologous species, such as sweetgum.
A further means of creating sterile trees is to introduce a transgene that specifically kills cells that are involved in floral development or production of gametes (genetic ablation). Numerous patents describe variations of genetic ablation for enabling controlled breeding of agricultural species, but they singly address either male or female sterility (Nasrallah et al. (1994); Tuttle and Crossland (1995); DeGreef et al. (1997); Mariani et al. (1998); Oliver et al. (1998)). One method includes delivering into the plant a gene encoding a cytotoxic substance associated with a male tissue specific promoter. Another method involves an antisense system in which a gene critical to fertility is identified and an antisense to the gene inserted in the plant. Some published patent applications also describe methods for complete sterility of plants (Mariani et al. (1988); Teasdale (1992); Bridges et al. (1990)). One method includes several cytotoxin encoding gene sequences, along with male tissue specific promoters and an antisense system. Another method uses xe2x80x9crepressorxe2x80x9d genes which inhibit the expression of another gene critical to male sterility. Additionally, it is yet to be convincingly demonstrated that these methods do not have a detrimental effect on vegetative development. Strauss and co-workers (1998) have seen marked reduction in growth of cottonwoods that contain several genetic ablation constructs whose expression was expected to be strictly limited to reproductive tissues.
An additional problem in genetic engineering of plants is the limited number of gene components such as introns or transcriptional terminators that are freely available for making synthetic genes. It has been noted that transgenes that have sequences duplicated elsewhere in the genome can be susceptible to transcriptional inactivation, possibly mediated by methylation of the repeated DNA. Therefore, multigene constructs run the risk of unstable expression if several of the genes contain repeated DNA because they share the same transcriptional terminator.
Thus, it is desired to identify sweetgum genes which are involved with flowering in order to derive promoter and/or enhancer and/or intron sequences for use in preparing transgenic plants or in order to interfere with normal flower development in transgenic sweetgum to produce sterile trees.
The present invention is directed to a gene and gene product of sweetgum (Liquidambar styraciflua), referred to herein as the LSAG gene, which is a homolog of the AGAMOUS gene, and to uses of the gene, gene product or parts of the gene.
In one aspect of the invention, the DNA and protein sequences are provided for sweetgum LSAG.
In a second aspect of the invention, constructs comprising at least a portion of an LSAG nucleic acid is provided for altering floral development. The constructs generally comprise a heterologous promoter, i.e., one not naturally associated with the LSAG gene, operably linked to the LSAG nucleic acid. The LSAG may be in sense or antisense orientation with respect to the promoter. Vectors containing the construct for use in transforming plant cells are also provided. Any plant cells can be transformed in accordance with the present invention. Preferred plant cells are plant cells of woody plants.
In a third apsect of the invention, plants having at least one cell transformed with the construct containing LSAG nucleic acid for altering floral development is provided. Such plants have a phenotype characterized by altered floral development. Preferred plants are woody plants.
In a fourth aspect of the invention, methods for producing plants having altered floral development are provided. The methods comprise the steps of transforming plant cells with a vector comprising at least a portion of an LSAG nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant exhibiting altered floral development.
In a fifth aspect of the invention, a promoter, an enhancer and an intron of the sweetgum LSAG gene are provided.
In a sixth aspect of the invention, gene constructs comprising the promoter and/or enhancer and/or intron of the LSAG gene and a heterologous gene are provided. Vectors containing these constructs are also provided. Plants having at least one cell containing these constructs are further provided by the invention.